Bivalent cations and amino-acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase Claire Vieille 1 , Kevin L. Epting 2 , Robert M. Kelly 2 and J. Gregory Zeikus 1 1 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA; 2 Department of Chemical Engineering, North Carolina State University, Raleigh, NC, USA Comparative analysis of genome sequence data from mesophilic and hyperthermophilic micro-organisms has revealed a strong bias against specific thermolabile amino- acid residues (i.e. N and Q) in hyperthermophilic proteins. The N þ Q content of class II xylose isomerases (XIs) from mesophiles, moderate thermophiles, and hyperther- mophiles was examined. It was found to correlate inversely with the growth temperature of the source organism in all cases examined, except for the previously uncharacterized XI from Bacillus licheniformis DSM13 (BLXI), which had an N þ Q content comparable to that of homologs from much more thermophilic sources. To determine whether BLXI behaves as a thermostable enzyme, it was expressed in Escherichia coli, and the thermostability and activity properties of the recombinant enzyme were studied. Indeed, it was optimally active at 70–72 8C, which is significantly higher than the optimal growth temperature (37 8C) of B. licheniformis. The kinetic properties of BLXI, determined at 60 8C with glucose and xylose as substrates, were comparable to those of other class II XIs. The stability of BLXI was dependent on the metallic cation present in its two metal-binding sites. The enzyme thermostability increased in the order apoenzyme , Mg 2þ –enzyme , Co 2þ –enzyme < Mn 2þ –enzyme, with melting tempera- tures of 50.3 8C, 53.3 8C, 73.4 8C, and 73.6 8C. BLXI inactivation was first-order in all conditions examined. The energy of activation for irreversible inactivation was also strongly influenced by the metal present, ranging from 342 kJ·mol 21 (apoenzyme) to 604 kJ·mol 21 (Mg 2þ – enzyme) to 1166 kJ·mol 21 (Co 2þ –enzyme). These results suggest that the first irreversible event in BLXI unfolding is the release of one or both of its metals from the active site. Although N þ Q content was an indicator of thermo- stability for class II XIs, this pattern may not hold for other sets of homologous enzymes. In fact, the extremely thermostable a-amylase from B. licheniformis was found to have an average N þ Q content compared with homologous enzymes from a variety of mesophilic and thermophilic sources. Thus, it would appear that protein thermostability is a function of more complex molecular determinants than amino-acid content alone. Keywords: Bacillus licheniformis; metal binding; thermo- stability; xylose isomerase. It has become apparent that protein thermostability arises not from a single chemical or physical factor, but from numerous subtle contributions integrated over the entire molecular structure [1 – 6]. Thermostable proteins usually exhibit no significant differences in backbone conformation when compared with less thermostable proteins, but they typically have increased numbers of salt bridges, side chain–side chain hydrogen bonds, and residues involved in a helices [7–9]. Stability at very high temperatures further requires that a particular enzyme resist thermally induced deleterious chemical reactions, which usually occur at insignificant rates at lower temperatures [10]. For example, one of the most evident patterns in the amino-acid composition of hyperthermophilic proteins is the bias against thermally labile amino-acid residues. This pattern is obvious on examination of the amino-acid compositions of the total protein content of eight mesophilic and seven hyperthermophilic micro-organisms for which genome sequence data are available (Table 1). The most striking difference is the 55% reduction in the number of glutamines in hyperthermophilic proteins; note also the 28% reduction in asparagines. As these two amino acids are easily deamidated at elevated temperatures [10– 13], it is not surprising that they are less abundant in proteins from hyperthermophiles. This observation raises the question of whether a relatively low N þ Q content is a signature of enhanced thermostability in proteins from mesophilic sources. The potential use of biocatalysts at high temperatures for technological purposes has drawn interest in developing thermoactive and thermostable enzymes that would provide significant processing advantages [14]. For example, thermostable xylose isomerases (XIs) (EC 5.3.1.5), which catalyze the isomerization of D-xylose to D-xylulose in vivo, are used for the conversion of D-glucose into D-fructose for the production of high-fructose corn syrup [15]. Elevated bioprocessing temperatures are preferred to achieve higher catalytic rates as well as more favorable equilibrium yields Correspondence to J. G. Zeikus, Department of Biochemistry and Molecular Biology, 410 Biochemistry Building, Michigan State University, East Lansing, MI 48824, USA. Fax: þ 517 353 9334, Tel.: þ 517 353 5556, E-mail: zeikus@msu.edu (Received 24 April 2001, revised 29 September 2001, accepted 10 October 2001) Abbreviations: XI, xylose isomerase; BLXI, Bacillus licheniformis xylose isomerase; DSC, differential scanning calorimetry. Eur. J. Biochem. 268, 6291–6301 (2001) q FEBS 2001 Table 1. Amino-acid content of the total proteins of selected mesophiles and hyperthermophiles. Calculations were performed using all the open reading frames described in the genomic sequences present in GenBank. Organism Ala Cys Asp Glu Phe Gly His Ile Lys Leu Met Asn Pro Gln Arg Ser Thr Val Trp Tyr Hyperthermophiles Aquifex aeolicus 5.90 0.79 4.32 9.63 5.13 6.75 1.54 7.32 9.40 10.57 1.92 3.60 4.07 2.04 4.91 4.79 4.21 7.93 0.93 4.13 Archaeoglobus fulgidus a 7.84 1.17 4.89 8.90 4.59 7.26 1.51 7.25 6.86 9.49 2.62 3.23 3.86 1.78 5.79 5.51 4.16 8.60 1.04 3.64 Aeropyrum pernix a 9.53 0.93 3.88 6.61 2.75 8.55 1.92 5.19 3.52 11.38 1.97 2.04 6.46 1.90 7.71 7.53 4.69 8.75 1.31 3.36 Methanococcus jannaschii a 5.54 1.27 5.52 8.67 4.20 6.41 1.43 10.45 10.36 9.38 2.33 5.24 3.38 1.44 3.85 4.46 4.06 6.85 0.71 4.33 Pyrococcus abyssi b 6.68 0.55 4.61 8.85 4.35 7.26 1.50 8.50 7.80 10.25 2.40 3.34 4.26 1.67 5.73 4.97 4.20 8.07 1.18 3.83 Pyrococcus horikoshii b 6.37 0.63 4.26 8.29 4.60 6.97 1.49 8.79 7.74 10.36 2.40 3.54 4.51 1.63 5.46 5.86 4.51 7.55 1.17 3.84 Thermotoga maritima 5.85 0.71 4.96 8.92 5.19 6.92 1.58 7.22 7.61 10.02 2.40 3.63 3.99 2.01 5.55 5.65 4.52 8.60 1.10 3.58 Average 6.82 0.86 4.63 8.55 4.40 7.16 1.57 7.82 7.61 10.21 2.29 3.52 4.36 1.78 5.57 5.54 4.34 8.05 1.06 3.82 Variance 1.72 0.06 0.25 0.77 0.57 0.4 0.02 2.31 4.02 0.4 0.06 0.75 0.84 0.04 1.15 0.88 0.05 0.4 0.03 0.09 Standard deviation 1.31 0.25 0.5 0.88 0.76 0.63 0.15 1.52 2.00 0.63 0.23 0.87 0.92 0.20 1.07 0.94 0.22 0.63 0.18 0.31 Mesophiles Bacillus subtilis 7.69 0.80 5.18 7.24 4.49 6.91 2.28 7.36 7.05 9.64 2.78 3.95 3.68 3.84 4.13 6.31 5.43 6.75 1.03 3.47 Campylobacter jejuni 6.79 1.22 5.27 7.02 5.99 5.80 1.64 8.66 9.48 10.77 2.23 6.29 2.67 3.06 3.05 6.43 4.05 5.25 0.65 3.67 Escherichia coli 9.55 1.11 5.20 5.91 3.87 7.42 2.26 5.95 4.48 10.56 2.86 3.88 4.41 4.42 5.58 5.67 5.35 7.11 1.48 2.83 Haemophilus influenzae 8.21 1.03 4.98 6.48 4.46 6.65 2.05 7.10 6.32 10.50 2.44 4.89 3.72 4.64 4.47 5.84 5.20 6.68 1.12 3.12 Helicobacter pylori 6.83 1.09 4.77 6.88 5.41 5.76 2.12 7.20 8.94 11.18 2.28 5.83 3.28 3.70 3.46 6.81 4.37 5.59 0.7 3.68 Neisseria meningitidis 10.5 1.44 5.19 6.12 4.06 7.86 2.17 5.72 5.57 9.76 2.46 3.95 4.14 3.90 5.38 5.38 5.58 6.71 1.18 2.94 Rickettsia prowazekii 6.05 1.10 4.83 5.78 4.88 5.42 1.91 10.88 8.38 10.10 2.17 6.65 3.15 3.14 3.39 6.76 5.21 5.59 0.72 3.89 Synechosystis 9.07 1.01 5.07 6.20 3.75 7.77 1.93 6.31 4.26 10.93 2.12 3.76 5.09 5.26 5.18 5.46 5.53 7.10 1.30 2.78 Average 8.09 1.10 5.06 6.45 4.61 6.70 2.04 7.40 6.81 10.43 2.42 4.90 3.77 3.99 4.33 6.08 5.09 6.35 1.02 3.30 Variance 2.06 0.03 0.03 0.26 0.53 0.80 0.04 2.49 3.49 0.27 0.07 1.25 0.52 0.49 0.84 0.28 0.28 0.49 0.08 0.16 Standard deviation 1.44 0.17 0.17 0.51 0.73 0.89 0.20 1.58 1.87 0.52 0.26 1.12 0.72 0.70 0.92 0.53 0.53 0.70 0.29 0.40 t Distribution Sigma 1.48 0.23 0.39 0.75 0.80 0.84 0.19 1.67 2.08 0.61 0.27 1.08 0.88 0.57 1.07 0.80 0.45 0.72 0.26 0.39 t 1.66 1.99 2.10 2 5.37 0.52 2 1.06 4.82 2 0.49 2 0.75 0.70 0.92 2.46 2 1.31 7.52 2 2.25 1.31 3.28 2 4.58 2 0.30 2 2.57 Confidence 5% x x x x x x x x (t 0.975 ¼ 2.16) Confidence 1% x x x x (t 0.995 ¼ 3.01) a Archaea in which Gln-tRNA is formed through the activities of GatDE and GatCAB; b Archaea in which Gln-tRNA is formed only through the activity of GatDE [54]. 6292 C. Vieille et al.(Eur. J. Biochem. 268) q FEBS 2001 [16]. Because of the commercial importance of XIs, their biochemical, biophysical, and structural properties have been extensively studied, and abundant sequence infor- mation is available [10,17–24]. On the basis of the absence or presence of a 50-residue insert at the N-terminus, XIs have been classified into class I and class II enzymes, respectively [25]. Two distinct metal-binding sites, M1 and M2, have been identified in all XIs: (a) the metal in site M1 is co-ordinated to four carboxylate groups; (b) the metal in site M2 is co-ordinated to one imidazole and three carboxylate groups. The metals in sites M1 and M2 were initially referred to as structural and catalytic metals, respectively [18,26,27], but these appellations are no longer valid, because later studies showed that both metals are directly involved in catalysis [24,28,29]. The stabilizing and activating metals are typically the bivalent cations Mg 2þ , Co 2þ , and Mn 2þ . Metal specificity depends on both the nature of the substrate (i.e. glucose or xylose) and whether the enzyme is a class I or class II XI. Thermus aquaticus XI, a class I enzyme, isomerizes glucose most efficiently when in the presence of Mn 2þ , but its activity toward xylose is highest with Co 2þ as the cofactor [26]. The class II Bacillus coagulans XI, on the other hand, isomerizes xylose most efficiently when in the presence of Mn 2þ , whereas its activity toward fructose is best promoted by Co 2þ [27]. Class I XIs are a relatively homogeneous group when it comes to thermostability, whereas class II XIs vary widely in this regard. This heterogeneity among class II XIs may arise from the existence of additional salt bridge(s) specific to thermostable class II XIs [30]. In a previous study of class II XIs, a positive correlation between the enzyme’s N þ Q content and the growth temperature of the source organism was observed: XIs from the most thermophilic sources typically had lower N þ Q content [23]. Of the XIs examined, only the enzyme from the mesophilic bacterium Bacillus licheniformis DSM13 (BLXI) was atypical. Originating from an organism that optimally grows at 37 8C, BLXI contains only 26 N þ Q residues, compared with 23 in the enzyme from the hyperthermophile Thermotoga neapolitana (optimal growth at 80 8C), and 46 in the XI from the mesophile Escherichia coli (optimal growth at 37 8C). The low Q (and, to some extent, N) content in proteins from hyperthermophiles (Table 1) raises an interesting question: does a relatively low N þ Q content in a protein from a mesophile indicate an unusually high thermostability for this protein? The location of N and Q residues within a protein structure is certainly a critical consideration, but detailed structural information is often not available to make this determination. In an attempt to test the simple hypothesis that class II XI stability at high temperatures correlates with its N þ Q content, independent of the growth temperature of the source organism, the biochemical and biophysical properties of the previously uncharacterized BLXI were determined. Particular empha- sis was placed on the influence of bivalent cations on activity and stability. Our results show that for class II XIs, N þ Q content relates to the enzyme’s functional temperature range and that BLXI thermostability is also directly related to the binding of specific metals as cofactors. At the same time, the simple relationship between thermostability and N þ Q content may not hold in general, as it is not the case for the thermostable a-amylase from B. licheniformis. MATERIALS AND METHODS B. licheniformis xylA gene cloning B. licheniformis strain DSM13 was grown at 37 8Cin Luria–Bertani broth [31]. A B. licheniformis genomic DNA library was constructed in vector pUC18 (Pharmacia, Piscataway, NJ, USA), using methods described in [23]. E. coli xyl – mutant HB101 (F – , hsdS20, ara-1, recA13, proA12, lacY1, galK2, rpsL20, mtl-1, xyl-5 ) [32] was transformed with the ligation mixture by electroporation and plated on M9 medium containing 0.2% xylose, 0.1% casamino acids, thiamine (500 mg·mL 21 ), and ampicillin (100 mg·mL 21 ). Only the transformants expressing a recombinant XI produced large colonies on this medium. Enzyme purification BLXI was purified from a 2-L culture of E. coli HB101 carrying plasmid pBL2 grown in M9 medium (comple- mented as above). After centrifugation for 5 min at 4000 g, the cell pellet was resuspended in 50 m M Mops (pH 7.0) containing 5 m M MgSO 4 and 0.5 mM CoCl 2 (buffer A). The cells were disrupted by two consecutive passes through a French pressure cell (American Instrument Co., Silver Spring, MD, USA), using a decrease in pressure of 96.5 MPa. After centrifugation at 25 000 g for 30 min, the supernatant was heat-treated at 60 8C for 10 min. The precipitated material was separated by centrifugation at 25 000 g for 30 min. The soluble fraction was loaded on to a DEAE–Sepharose Fast-Flow column equilibrated with buffer A. The protein was eluted with a linear 0.05– 0.4 M NaCl gradient in buffer A. The active fractions were analyzed by SDS/PAGE (12% acrylamide) and stained with Coomassie blue R250. The homogeneous fractions were pooled and extensively dialyzed against buffer A. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad, Richmond, CA, USA), with BSA as the standard. The purified enzyme was stored at 2 70 8C until use. Molecular mass determination BLXI molecular mass was determined by gel filtration using a Sephacryl S-300 HR column (1.4 cm £ 160 cm) calibrated with blue dextran and protein standards of 443, 200, 150, and 66 kDa (Sigma Chemical Co., St Louis, MO, USA). The flow rate was 0.2 mL·min 21 . EDTA treatment The purified enzyme was incubated overnight at 4 8Cin buffer A containing 10 m M EDTA. It was then dialyzed twice against 50 m M Mops (pH 7.0) (SigmaUltra, Sigma Chemical Co.) containing 2 m M EDTA, and finally dialyzed twice against 50 m M Mops (pH 7.0), this time without EDTA. The apoenzyme was divided into aliquots and stored at 2 70 8C until use. Enzyme assays BLXI activity was assayed routinely with glucose as the substrate. The enzyme (0.06 mg·mL 21 ) was incubated in q FEBS 2001 B. licheniformis xylose isomerase (Eur. J. Biochem. 268) 6293 50 mM Mops (pH 7.0 at room temperature) containing 1m M CoCl 2 and 1 M glucose at 60 8C for 20 min The reaction was stopped by transferring the tubes to an ice bath. The amount of fructose produced was determined by the cysteine/carbazole/sulfuric acid method [33]. To determine the effect of temperature on BLXI activity, the holo-BLXI was incubated in the reaction mixture at the temperatures of interest in a Perkin–Elmer Cetus GeneAmp PCR system 9600 (Perkin – Elmer, Norwalk, CT, USA) for 20 min. To determine the kinetic parameters, assays were performed in the presence of either 80–1400 m M glucose or 20–900 mM xylose. The amounts of fructose and xylulose produced were determined using the cysteine/carbazole/sulfuric acid method. Absorbance was measured at 537 nm and 560 nm for xylulose and fructose, respectively. One unit of isomerase activity is defined as the amount of enzyme that produces 1 mmol of product per min under the assay conditions. Activation of apo-BLXI by metals To examine the effect of bivalent cations on enzyme activity, CoCl 2 , MnCl 2 , or MgCl 2 were added to the apoenzyme reaction mixture at concentrations of 0.003– 100 m M (activity on glucose) or 0.002– 0.03 mM (activity on xylose). Activity on xylose was determined using 0.024 mg·mL 21 apo-BLXI and 635 mM xylose. Activity on glucose was determined using 0.06 mg·mL 21 apo-BLXI and 1 M glucose. The activation constant, K act , for a metal is defined as the metal concentration that results in 50% of maximum activity [34,35]. Enzyme thermoinactivation The apo-BLXI (0.17 mg·mL 21 )in50mM Mops (pH 7.0 at room temperature) was incubated at various temperatures (in a Perkin –Elmer Cetus GeneAmp PCR system 9600) for different periods of time. Thermoinactivation was stopped by transferring the tubes to an ice bath. Residual activity was determined under the conditions described above, except that the reaction mixture contained 5 m M CoCl 2 instead of 1m M CoCl 2 . To determine the effect of metals on the apoenzyme stability, 0.5 m M CoCl 2 , 0.5 mM MnCl 2 ,or 2m M MgCl 2 was added to the enzyme solution. The resulting solution was equilibrated for 30 min at 30 8C before thermoinactivation was initiated (preincubation conditions known to be sufficient for the metal to reach equilibrium between the buffer and enzyme metal-binding sites [19]). Heat-induced enzyme precipitation Heat-induced enzyme precipitation was monitored from 25 8Cto908C by light scattering (l ¼ 580 nm), using the apo-BLXI (0.08 mg·mL 21 )in10mM Mops (pH 7.0). Absorbance measurements were conducted in 0.3-mL quartz cuvettes (pathlength 1 cm) using a Beckman DU-650 spectrophotometer equipped with a Peltier cuvette-heating system. The increasing thermal gradient was 1.0 8C·min 21 . The effect of metals on apo-BLXI precipitation was studied in the presence of 0.5 m M CoCl 2 , 0.5 m M MnCl 2 ,or5mM MgCl 2 . PH studies The effect of pH on BLXI activity was determined at 64 8C using the routine assay described above, except that the Mops buffer was substituted with 100 m M sodium acetate (pH 4.0–5.7), 100 m M Pipes (pH 6.0–7.5), or 100 mM Hepps (pH 7.5–8.7). All pHs were adjusted at room temperature, and the DpK a /DT values for acetate, Pipes, and Hepes (0.000, 2 0.0085, and 2 0.011, respectively) [36] were taken into account for the results. Differential scanning calorimetry (DSC) DSC experiments were performed on a Nano-Cal differen- tial scanning calorimeter (Calorimetry Sciences Corp., Provo, UT, USA) using a scan rate of 1 8C·min 21 . Samples were scanned from 25 8C to 100 8C. The apoenzyme (obtained by EDTA treatment, see above) was scanned against 50 m M Mops (pH 7.0). For the metal-containing enzymes, the apoenzyme was incubated for 2 h at room temperature with 5 m M metal chloride. The enzyme solution was then dialyzed once against 1 L 50 m M Mops (pH 7.0) to remove the metal that was not tightly bound to the enzyme. Each metal-containing enzyme was scanned against the corresponding dialysis buffer. The dialysis buffer was used to generate the baseline. The enzyme containing both Mg 2þ and Co 2þ was dialyzed against buffer A, then scanned against the dialysis buffer as control. RESULTS AND DISCUSSION Cloning of the B. licheniformis xylA gene Plasmid pBL1 was characterized from an HB101 transfor- mant that formed large colonies on M9 medium containing xylose. Comparison of the physical map of the plasmid pBL1 insert with that of plasmid pWH1450 [37] indicated Fig. 1. Determination of BLXI molecular mass by gel filtration. V e /V o is the ratio of a protein’s elution volume to the elution volume of blue dextran. (X) Protein standards; (A) BLXI. Linear regression (1), with an r 2 of 0.951, is based on the elution data of all four protein standards. Linear regression (2), with an r 2 of 0.981, is based on the elution data of the 200, 150, and 66 kDa protein standards. Linear regressions (1) and (2) give molecular masses of 200 and 177.5 kDa, respectively, for BLXI. 6294 C. Vieille et al.(Eur. J. Biochem. 268) q FEBS 2001 that pBL1 contained B. licheniformis xylR, xylA, and a truncated xylB. A 1.2-kb Sph I–Eco RI fragment was deleted from the pBL1 insert to inactivate the B. licheniformis xylR repressor gene, leading to plasmid pBL2. Plasmid pBL2 was used to express BLXI in the rest of the study. Purification of the recombinant protein and physical properties The B. licheniformis xylA gene was expressed from its own promoter in plasmid pBL2. The recombinant enzyme was purified (heat treatment plus DEAE–Sepharose chromato- graphy) from an HB101 (pBL2) 2-L culture grown in M9 medium plus xylose. The purified enzyme was shown to be homogeneous by SDS/PAGE and staining with Coomassie blue. Approximately 100 mg enzyme was obtained from the 2-L culture. A molecular mass of 200 kDa for the native protein was estimated by gel filtration (Fig. 1). Analysis by SDS/PAGE showed a single band with a molecular mass of about 50 kDa. This estimate is in agreement with that expected from the protein sequence (50 905 Da). These results indicate that BLXI is expressed as a homotetramer in E. coli. It is interesting to note that XIs from the thermophile Thermoanaerobacterium thermosulfurigenes and the hyperthermophile T. neapolitana, both homotetra- mers in their native forms, are expressed as active dimers in E. coli [38]. Effects of temperature and pH on BLXI activity The effect of temperature on BLXI activity was determined by measuring the holoenzyme activity on glucose in the presence of 1 m M CoCl 2 . As shown in Fig. 2A, BLXI is optimally active between 70 8C and 72 8C. Above 72 8C, the enzyme rapidly loses activity, and it is completely inactive at 80 8C. The Arrhenius plot for BLXI activity is linear between 35 8C and 66 8C. The estimated energy of activation (E a ) for BLXI activity is 76 kJ·mol 21 ,anE a value comparable to those for E. coli and T. neapolitana XI activities (70 and 80 kJ·mol 21 , respectively; unpublished data). The effect of pH on BLXI activity was determined by measuring the holoenzyme activity on glucose between pH 4.8 and 8.2 (values after temperature correction). BLXI is optimally active at pH 7.2 and shows more than 80% activity between pH 6.8 and 7.6 (Fig. 2B). BLXI kinetic parameters BLXI kinetic parameters were determined at 60 8C for glucose and xylose using the holoenzyme in the presence of 1m M CoCl 2 (Table 2). Not surprisingly, BLXI is about six times more efficient on xylose than on glucose, as indicated by the values of V max /K m . Compared with other type II thermophilic XIs (Table 2), BLXI has average kinetic parameters, but it has a relatively low catalytic efficiency on xylose. BLXI thermostability and inactivation characteristics BLXI thermostability was first characterized using the holoenzyme in the presence of 1 m M CoCl 2 . In these conditions, BLXI has a half-life of 14 h at 64 8C, 70 min at 67 8C, and 2 min 40 s at 70 8C. Figure 3 shows that BLXI inactivation at 68 8C was first-order. Inactivation rates obtained for three different enzyme concentrations at 68 8C Table 2. Kinetic constants of thermophilic type II XIs. Organism T (8C) Glucose Xylose Reference V max (U·mg 21 ) K m (mM) V max /K m V max (U·mg 21 ) V max /K m K m (mM) B. licheniformis DSM13 60 7.7 145 0.053 22.2 67 0.33 This work B. stearothermophilus 60 6.0 220 0.027 44.5 100 0.44 [55] T. saccharolyticum B6A 65 6.3 120 0.053 17.6 16 1.10 [56] T. thermosulfurigenes 4B 65 5.3 142 0.037 15.7 20 0.78 [56] T. maritima DSM 3109 90 16.2 118 0.137 68.4 74 0.92 [57] T. neapolitana 5068 90 22.4 89 0.253 52.2 16 3.28 [23] Fig. 2. Effects of temperature and pH on BLXI specific activity. (A) Arrhenius plot of BLXI specific activity as a function of temperature. The linear regression was only applied to the temperature points below the optimum temperature for activity. (B) Effect of pH on BLXI activity. Assays were performed at 64 8Cin 100 m M sodium acetate (A; pH 4.0– 5.7), 100 mM Pipes (X; pH 6.0 –7.5), or 100 mM Hepes (K pH 7.5 –8.7). The DpK a /DT values for acetate, Pipes, and Hepps were taken into account to calculate the pH values at 64 8C. All assays were performed in triplicate. q FEBS 2001 B. licheniformis xylose isomerase (Eur. J. Biochem. 268) 6295 (Fig. 3A; 0.012, 0.01, and 0.007 min 21 at 0.05, 0.5, and 2.5 mg·mL 21 enzyme, respectively) indicate that BLXI inactivation is slightly dependent on concentration, with BLXI stability increasing with enzyme concentration. At 0.5 and 2.5 mg·mL 21 , enzyme precipitation was noted during BLXI inactivation. Residual activities were compared in whole-enzyme (sample before centrifugation) and soluble (supernatant after centrifugation) fractions after BLXI inactivation at 68 8C. The soluble fraction showed an inactivation rate that was slightly higher than the whole enzyme (Fig. 3B). This difference does not appear to be significant. Inactivation was accompanied by heavy aggregation at this enzyme concentration, and the difference in inactivation rate between the two samples probably results from the trapping of some active soluble enzyme molecules in the aggregate. As the aggregate increased in size with inactivation time, more and more soluble enzyme may remain trapped in the insoluble pellet during centrifugation. These results suggest that the precipitated enzyme was completely inactive, and that the soluble fraction remained fully active. The Arrhenius plot of BLXI inactivation rates was linear (not shown) with an E a of 908 kJ·mol 21 . This estimate is consistent with an inactivation mechanism that involves significant unfolding [10]. Metal requirement for BLXI activity To determine which metal cation (Co 2þ ,Mn 2þ ,orMg 2þ ) best activates BLXI on glucose and on xylose, the apoenzyme activity was tested on both sugars in the presence of increasing concentrations of each cation (in the chloride form; Fig. 4). In the absence of metal, the apoenzyme was completely inactive on both substrates. Co 2þ was by far the best activating cation for BLXI activity on glucose. BLXI activity in the presence of Mn 2þ and Mg 2þ reached only 6% and 15%, respectively, of the activity in the presence of Co 2þ (Fig. 4A). Co 2þ ,Mn 2þ , and Mg 2þ activation constants for BLXI activity on glucose are approximately 0.5 m M, 0.3 mM, and 4.5 mM, respectively. The metal requirement of BLXI for activity on xylose was significantly different from its metal requirement for activity on glucose. The three metals studied stimulate BLXI activity on xylose in the order Mn 2þ $ Co 2þ . Mg 2þ (Figs 4B,C). Activity in the presence of Mg 2þ was one-half the activity in the presence of Mn 2þ . K act values of Co 2þ , Mn 2þ ,andMg 2þ forBLXIactivityonxyloseare < 0.01 m M, 0.0075 mM, and 0.2 mM, respectively, 22–50 times lower than for BLXI activity on glucose. Marg & Clark [27] obtained a similar result with B. coagulans XI: K act values were 10 times higher for glucose than for xylose, and the relative effectiveness of the three metals was the same. Occupancy rate of the M2 site necessary for activity on glucose may be higher than that for activity on xylose; it has been found that some XIs are active on xylose with only the M1 site occupied [27]. Also, the metal-specific Fig. 3. Characteristics of holo-BLXI inactivation at 68 8C. Assays were performed in triplicate. All linear regression had correlation coefficients r 2 above 0.96. (A) Effect of enzyme concentration on holo- BLXI inactivation rate. Enzyme concentrations: (A) 0.05 mg·mL 21 ; (X) 0.5 mg·mL 21 ;(K) 2.5 mg·mL 21 . Inactivation rates corresponding to the slopes of the linear regressions for the three inactivation curves were 0.012 min 21 at 0.05 mg·mL 21 , 0.01 min 21 at 0.5 mg·mL 21 , and 0.007 min 21 at 2.5 mg·mL 21 . (B) Remaining activity in the total and soluble holo-BLXI fractions. Initial enzyme concentration was 2.5 mg·mL 21 . The soluble fraction corresponded to the supernatant of the whole enzyme fraction, after centrifugation. (K) Whole enzyme; (O) soluble fraction. Fig. 4. Apo-BLXI activation by Co 21 (A), Mn 21 (X), and Mg 21 (S). (A) Apo-BLXI specific activity with glucose as the substrate. (B) and (C) Apo-BLXI specific activity with xylose as the substrate. Assays were performed in triplicate. 6296 C. Vieille et al.(Eur. J. Biochem. 268) q FEBS 2001 differences observed in enzyme–metal binding are probably related to differences between the metals with respect to co-ordination geometries. These differences may influence the metal preferences for glucose as opposed to xylose isomerization. For example, whereas Mn–BLXI was highly active on xylose, it was barely active on glucose. Metal requirement of BLXI for stability To determine the metal requirement of BLXI for thermostability, the apoenzyme was incubated in the presence of 0.5 m M Co 2þ , 0.5 mM Mn 2þ ,or2mM Mg 2þ at different temperatures and for various periods of time. The metal was allowed to equilibrate between the buffer and the enzyme by preincubating the enzyme–metal mixture at 30 8C for 30 min. Remaining activity was measured with glucose as the substrate in the presence of 4 m M CoCl 2 . The apoenzyme was significantly less stable than the enzyme containing Co 2þ ,Mn 2þ ,orMg 2þ (Fig. 5 and Table 3). Of the three metal cations, Mn 2þ stabilized BLXI best. Co 2þ was only slightly less stabilizing than Mn 2þ (Table 3). Mg 2þ was significantly less efficient than the other two metals at stabilizing BLXI. This metal-specific protection of BLXI against inactivation is very similar to the situation observed with class I XIs: Mg 2þ does not protect type I XIs from unfolding to the extent that Co 2þ does [39,40]. The E a values of BLXI inactivation were calculated for the apoenzyme in the presence and absence of metals (Table 3). The nature of the metal present clearly affected the E a of BLXI inactivation. It was remarkably high in the presence of Co 2þ or Mn 2þ , which explains why BLXI specific activity decreased rapidly above 72 8C (Fig. 2). As both the E a of inactivation and the temperature at which the enzyme starts inactivating at a measurable rate increase from the apoenzyme to the Mn 2þ -containing enzyme, loss of the metal cofactor could be the limiting step in BLXI inactivation. Higher E a values of inactivation for the Co 2þ – enzyme and the Mn 2þ –enzyme reflect the higher thermal energy that these metal–enzyme complexes can accumulate before losing the tightly bound metal, causing them to unfold. The higher E a of inactivation provided by Co 2þ and Mn 2þ compared with that provided by Mg 2þ reflects the different binding affinities of these cations for BLXI. The extremely high E a of BLXI inactivation in the presence of Co 2þ or Mn 2þ are interesting. With a similar inactivation mechanism, E a of inactivation of T. thermo- sulfurigenes XI is only 490 kJ·mol 21 in the presence of Fig. 5. Arrhenius plots of apo-BLXI inactivation rates in the absence (K) or presence of 0.5 m M Co 21 (A), 0.5 mM Mn 21 (X), or 2m M Mg 21 (S). All linear regression had correlation coefficients r 2 above 0.99. Fig. 6. Determination of apo-BLXI precipitation temperature in the presence of 2 m M Mg 21 (S), 0.5 mM Co 21 (A), or 0.5 mM Mn 21 (X). Table 3. Effect of metals on apo-BLXI stability. NI, Data not interpretable. Metal Kinetic stability Thermodynamic stability: Half-life (min) E a of inactivation (kJ·mol 21 ) Precipitation temperature (8C) Melting temperature (8C) No metal 24 (at 40 8C) 342 NI 50.3 2m M MgCl 2 53 (at 54 8C) 604 57.1 53.3 35 (at 56 8C) 0.5 m M CoCl 2 53 (at 69.5 8C) 1166 69.9 73.4 18 (at 70.5 8C) 0.5 m M MnCl 2 50 (at 71.5 8C) 1073 72.8 73.6 16 (at 72.5 8C) q FEBS 2001 B. licheniformis xylose isomerase (Eur. J. Biochem. 268) 6297 Co 2þ (C. Vieille & J. G. Zeikus, unpublished results). Structural information for BLXI in the presence of Mn 2þ (or Co 2þ ) and Mg 2þ would provide insight into the molecular determinants responsible for the differences in E a . The large variation in E a of BLXI inactivation in the presence of different metals suggests that the differences in E a of inactivation for BLXI and T. thermosulfurigenes XI are related to differences in metal co-ordination geometries. Figure 6 and Table 3 show the effect of metals on BLXI precipitation temperature. This temperature increased in the order Mg 2þ –BLXI , Co 2þ –BLXI , Mn 2þ –BLXI. The precipitation temperatures in the presence of metals correlate well with the inactivation data (Table 3). Precipitation experiments with the apoenzyme did not provide reproducible data (not shown). The melting temperature for BLXI was determined by DSC in the presence and absence of metals (Fig. 7 and Table 3). It increased in the order apo-BLXI , Mg 2þ – BLXI , Co 2þ –BLXI , Mn 2þ –BLXI , (Mg 2þ þ Co 2þ )–BLXI. These values are consistent with BLXI inactivation and precipitation temperatures. The lower stability and lower E a of inactivation of BLXI in the presence of Mg 2þ reflect the fact that the enzyme binds Mg 2þ with lower specificity (the K act of Mg 2þ is 10 times higher that that of Co 2þ or Mn 2þ for both glucose and xylose). However, the influence of pH on the decreased protection of BLXI by Mg 2þ against thermoinactivation needs to be considered. The metals bind the enzyme through one His imidazole and several carboxylate groups. By affecting the carboxylate and His imidazole pK a values, pH affects XI –metal binding. It has been shown that Mg 2þ optimally binds XIs at pH values higher than either Co 2þ or Mn 2þ [41]. Whereas Co 2þ and Mn 2þ are present in both metal sites in crystals of Arthrobacter XI at pH 6.0, crystals of Arthrobacter XI do not contain any Mg 2þ at pH 6.0, and contain Mg 2þ only in site M1 at pH 8.0 [42]. As all Fig. 7. Thermal unfolding of apo-BLXI in the presence and absence of metals followed by DSC. See Materials and methods for experimental details. Fig. 8. Distribution of the N 1 Q (A), Q (B), and N (C) contents in the 90 B. licheniformis proteins of known sequence. Sequences were obtained from GenBank. 6298 C. Vieille et al.(Eur. J. Biochem. 268) q FEBS 2001 inactivation experiments in this work were performed at pH 7.0, Mg 2þ binding was probably not optimal. The difference in BLXI stabilization provided by Mg 2þ v Co 2þ or Mn 2þ may be smaller if BLXI inactivation were tested at a higher pH. The significance of cation binding in XI stability has not yet been examined closely. However, some information on this issue is available. Site-directed mutagenesis has been used to partially fill a metal-binding site with the side chain of an amino acid. These mutations to both metal-binding sites, M1 and M2, resulted in destabilized XIs. For example, mutation of His220 in the class I S. rubiginosus XI affected metal binding at M2, which in turn was responsible for destabilization [35]. A similar observation has been made for the class II E. coli XI: mutation of His271 (ligand to metal 2) significantly destabilized the enzyme without changing its structure appreciably (as determined by CD analysis) [43]. Other point mutations triggering confor- mational changes in active-site residues have also been found to destabilize XIs [44], suggesting that thermal unfolding starts through movements of active-site residues. Metal cations probably act to lock the active site in a stable conformation, which is lost as soon as the metal leaves the active site. Specific differences in stabilization efficacy between metals may be due to their ability to adopt different geometries in the same site in the absence of substrate. In the crystal structures of the S. rubiginosus enzyme, for example, Co 2þ in site 1 is tetra-co-ordinated or penta-co- ordinated and it adopts a strongly distorted geometry [45,46], whereas Mn 2þ is hexa-co-ordinated and adopts an octahedral geometry [18]. N 1 Q content as a general indicator of protein thermostability in mesophiles We reported previously [23] that the N þ Q content of class II XIs correlated with the growth temperature of the source organism, with the notable exception of BLXI. It is also interesting that of the B. licheniformis proteins with sequence available, the Q and N þ Q contents in BLXI are among the lowest (Fig. 8A,B). This is not the case, however, for the N content of BLXI (Fig. 8C). The entire genome of several mesophilic and hyperthermophilic organisms were analyzed in recent studies [9,47–49]. All these studies reported a decrease in the content of uncharged polar amino acids (i.e. Q, N, S, and T) and an increase in charged amino-acid residues (i.e. K, E, and R) in hyperthermophilic proteins. As S and T can catalyze the deamination and backbone cleavage of Q and N residues [48,50], a reduction in all four of these residues would minimize deamination. Although our prediction that BLXI, based on its low N þ Q content, had thermophilic properties (i.e. high thermostability and optimal activity at high temperatures) proved to be correct, a high N þ Q content does not necessarily predict that an enzyme will be thermolabile. This observation is evident from Fig. 8: the B. licheniformis a-amylase, which has an N þ Q content higher than the average (4.88% N, 4.30% Q), is an extremely thermostable enzyme, with optimal activity at 90 8C [51]. In addition, the N and Q contents of B. licheniformis a-amylase are close to the average N and Q contents (5.04 ^ 1.25% and 3.77 ^ 1.03%, respectively) of 20 homologous a-amylases from micro-organisms with differing growth temperature optima (data not shown). It is interesting to note, however, that five out of the seven thermostabilizing mutations that have been identified in this a-amylase (by Declerk et al. [52,53]) are substitutions of N or Q residues by less thermolabile amino acids. Thus, it appears that the N þ Q content alone does not account for thermostability, and that the location of these residues in the protein’s 3D structure must be taken into account. ACKNOWLEDGEMENTS This work was supported by the US National Science Foundation, grants NSF-Bes-9809964 (MSU) and NSF-Bes 9817067 (NCSU). We express our deep gratitude to Dr A. Roy Day for his help with the statistical analysis in Table 1 and Christopher B. Jambor for editing the manuscript. REFERENCES 1. 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The class II Bacillus coagulans XI, on the other hand, isomerizes xylose most efficiently